U.S. patent application number 11/988440 was filed with the patent office on 2009-06-11 for exosome ligands, their preparation and uses.
Invention is credited to Alain Delcayre, Jean-Bernard Le Pecq.
Application Number | 20090148460 11/988440 |
Document ID | / |
Family ID | 37670969 |
Filed Date | 2009-06-11 |
United States Patent
Application |
20090148460 |
Kind Code |
A1 |
Delcayre; Alain ; et
al. |
June 11, 2009 |
EXOSOME LIGANDS, THEIR PREPARATION AND USES
Abstract
The present invention relates to exosome-specific ligands and
compositions comprising the same. The invention also relates to
methods of generating said ligands and compositions, to methods of
using said ligands or compositions, e.g., to block the exosome
pathway or to detect and/or characterize exosomes in a sample or
subject, as well as to the antigens contacted by said ligands or
compositions. The application can be used in experimental,
research, therapeutic, prophylactic or diagnostic areas.
Inventors: |
Delcayre; Alain; (San Jose,
CA) ; Le Pecq; Jean-Bernard; (Menlo Park,
CA) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
37670969 |
Appl. No.: |
11/988440 |
Filed: |
July 7, 2006 |
PCT Filed: |
July 7, 2006 |
PCT NO: |
PCT/IB2006/002907 |
371 Date: |
January 8, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60697376 |
Jul 8, 2005 |
|
|
|
Current U.S.
Class: |
424/152.1 ;
424/178.1; 506/9; 514/19.4; 514/2.4; 514/3.8; 514/4.3;
530/388.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 39/0012 20130101; A61P 37/00 20180101; A61P 31/04 20180101;
A61P 31/14 20180101; C07K 2317/56 20130101; A61P 37/06 20180101;
A61P 31/18 20180101; C07K 16/18 20130101; A61P 31/00 20180101 |
Class at
Publication: |
424/152.1 ;
514/8; 424/178.1; 530/388.2; 506/9 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/17 20060101 A61K038/17; A61K 39/00 20060101
A61K039/00; C07K 16/18 20060101 C07K016/18; C40B 30/04 20060101
C40B030/04; A61P 35/00 20060101 A61P035/00; A61P 31/18 20060101
A61P031/18; A61P 31/14 20060101 A61P031/14; A61P 31/04 20060101
A61P031/04; A61P 37/06 20060101 A61P037/06 |
Claims
1-38. (canceled)
39. A method for neutralizing exosomes in a subject, the method
comprising administering to a subject in need thereof a
neutralizing-effective amount of an exosome-specific ligand, said
administration causing a neutralization of exosomes.
40. The method of claim 39, wherein the exosome-specific ligand is
an antibody or a fragment or derivative thereof.
41. The method of claim 40, wherein the antibody is a monoclonal
antibody.
42. The method of claim 40, wherein the antibody, or fragment or
derivative thereof, comprises a heavy chain variable polypeptide
sequence encoded by a nucleotide sequence selected from SEQ ID NOs:
1 to 12.
43. The method of claim 39, wherein the exosome-specific ligand is
a protein, or a domain thereof, that selectively binds an
exosome-specific marker.
44. The method of claim 43, wherein the protein is lactadherin or a
fragment thereof comprising at least the C1 and/or C2 domain.
45. The method of claim 39, wherein the exosome-specific ligand is
a chimeric polypeptide comprising an exosome-specific
ligand-binding domain fused to a dimerization or multimerization
domain, in particular to an Fc portion of an immunoglobulin.
46. The method of claim 39, wherein the exosome-specific ligand
comprises several exosome-specific ligand-binding domains.
47. The method of claim 46, wherein the exosome-specific ligand
comprises a protein, or a domain thereof, that selectively binds an
exosome-specific marker, fused to an exosome-specific antibody or a
fragment or derivative thereof.
48. A method for neutralizing a pathological immune response in a
subject, the method comprising administering to a subject in need
thereof a neutralizing-effective amount of an exosome-specific
ligand, said administration causing a neutralization of said
pathological immune response.
49. The method of claim 48, wherein said pathological immune
response is selected from an allergic response, an inflammatory
response, including host versus graft response, and an autoimmune
response.
50. A method for neutralizing or treating (viral or microbial)
infection in a subject, the method comprising administering to a
subject in need thereof an amount of an exosome-specific ligand
effective to neutralize exosomes in said subject, said
administration causing a neutralization or treatment of said
infection.
51. The method of claim 50, wherein the virus is selected from an
immunodeficiency virus and a hepatitis virus.
52. A method for treating cancer (e.g., by reducing cancer immune
evasion) in a subject, the method comprising administering to a
subject in need thereof an amount of an exosome-specific ligand
effective to neutralize exosomes in said subject, said
administration causing a treatment of said cancer.
53. A composition comprising an exosome-specific ligand and a
pharmaceutically acceptable carrier or excipient.
54. An antibody, or a fragment or derivative thereof having
substantially the same antigen specificity, wherein said antibody
selectively binds an exosome.
55. The antibody, or fragment or derivative thereof, of claim 54,
which comprises a heavy chain variable polypeptide sequence encoded
by a nucleotide sequence selected from SEQ ID NOs: 1 to 12.
56. A method of producing exosome-specific antibodies, comprising:
1) preparing exosomes from a non-human animal with a given genetic
background; 2) immunizing a non-human animal with said vesicles,
wherein animals for immunization and preparation of vesicles share
the same or very similar genetic background; and 3) collecting,
from the immunized animal, antibodies reacting with the vesicles of
step 1 or corresponding antibody-producing cells.
57. A method of isolating exosome-specific ligands, comprising: 1)
preparing exosomes, 2) screening libraries of compounds reacting
with said vesicles, 3) selecting exosome ligands from step 2 that
do not react with the exosome-producing cell.
58. A method of stimulating B cells in vivo, comprising
administering to an animal or a subject in need thereof an amount
of exosomes sufficient to cause a stimulation of B cells in said
animal or subject.
Description
[0001] The present invention relates to exosome-specific ligands
and compositions comprising the same. The invention also relates to
methods of generating said ligands and compositions, to methods of
using said ligands or compositions, e.g., to block the exosome
pathway or to detect and/or characterize exosomes in a sample or
subject, as well as to the antigens contacted by said ligands or
compositions. The invention can be used in experimental, research,
therapeutic, prophylactic or diagnostic areas.
BACKGROUND
[0002] Since their discovery, a growing number of therapeutic
applications are in development using exosomes derived from various
producing cells, such as dendritic cells (DC), T lymphocytes, tumor
cells and cell lines (1,2). For instance, DC-derived exosomes (also
designated dexosomes) pulsed with peptides derived from tumor
antigens elicit anti-tumor responses in an animal model for the
matching tumor (3). Two Phase-I clinical trials using autologous
dexosomes for the treatment of lung cancer (4) and melanoma (5),
respectively, have recently been completed.
[0003] Exosomes derived from tumor cells, cell lines, T cells, are
also being assessed as an alternative to dexosomes for the
preparation of cancer vaccines (6-9). However, this approach was
recently challenged by findings suggesting that these non-DC
derived vesicles could also induce immune tolerance (10,11), T cell
apoptosis (12,13), metastasis or angiogenesis (14). Given that the
protein composition of exosomes depends on the nature of the
producing cells, the biological function of exosomes from diverse
origin is expected to vary. However, the absence of a standardized
and specific method of exosome preparation between laboratories as
well as of common exosomes-specific quality control methods to
guaranty the classification of vesicles as true exosomes has
complicated the interpretation of experimental findings. Indeed,
since cells are producing vesicles through various pathways (15),
the possibility of contamination of exosomes by other vesicles and
the plausible confusion of exosome properties with that, for
instance, of vesicles shed from the cell surface (10,16) exist.
[0004] Currently, product characterization and quality control for
Dexosome therapy relies in part on biomarkers such as CD81 or HLA
Class I and II that are common to various biological objects (2).
Indeed, these markers are also found on the cell surface as well as
microparticles and apoptotic bodies. Clearly, the provision of
reagents specific for biomarkers that are unique to exosomes would
be a significant improvement for product characterization and QC
purposes of this emerging new therapeutic agent. Such reagents
would enable to assess and compare the quality and purity of
exosomes prepared from various cell types and in different
laboratories. They would also be helpful to establish more
accurately and definitely the biological functions of true
exosomes, as well as to neutralize exosomes in pathological
conditions.
[0005] Indeed, new studies are now revealing that exosomes may play
a critical role in some pathophysiological situations and therefore
these vesicles are now also emerging as potential drug targets. In
particular, exosomes released by retrovirus-infected and cancer
cells display modified properties that could lead to immune evasion
of viruses such as HIV and HCV as well as tumors. The most
remarkable recent result concerns the hijacking of the exosome
pathway by retroviruses. A series of experiments strongly suggest
that HIV can not only bud from the plasma membrane in T cells to
generate infectious viral particles, but can also bud in
macrophages from the limiting membrane of the MVB (17,18, 18b,
18c). This would allow the viruses to enter the exosome pathway. In
this model, a viral capside could become inserted inside an exosome
and be released by the cell while totally hidden in a vesicle
resistant to complement-induced lysis, and be transferred directly
and rapidly to other APC. The exosome with its HIV stowaway becomes
a diabolical "Trojan Horse", which, instead of being targeted for
elimination by the immune system, takes advantage of its components
to spread in disguise and safely multiply. As mentioned in (18b),
such exosomes are stable and infectious. Furthermore, this system
could be used by other retroviruses and could even allow the spread
of retrotransposons (18). Other findings suggest that human
hepatitis C virus (HCV) could also use a similar strategy (19). In
this case, the exosome might represent the unique mode of spreading
for the virus (20). Other viruses like Epstein-Barr could direct
viral proteins to exosomes to alter their properties and decrease
the immune response (21,22). Similarly, exosomes derived from tumor
cells may have altered properties and functions (10). Like virally
infected cells, tumor cells can release modified exosomes by
incorporating specific proteins (23). By doing so, if these first
interesting observations were generalized, tumor exosomes could
become a new important component for the understanding of cancer
pathophysiology. They could prevent the transfer of antigens to DC
causing immunotolerance, transfer to neighbouring cells various
proteins such as angiogenic factors to favour their own growth.
[0006] Accordingly, it would be particularly useful to develop
therapeutic drugs that specifically block the exosome pathway in
situation where the pathway leads, for instance, to virus
propagation or tumor immune evasion and growth. In addition,
exosome-specific ligands or reagents also represent much needed
experimental, research and diagnostic tools for the accurate
characterization of exosomes and their drug derivatives.
SUMMARY OF THE INVENTION
[0007] The present invention now discloses exosome-specific ligands
or reagents, methods for generating such ligands or reagents, as
well as their uses, e.g., as therapeutic drugs to block the exosome
pathway or as diagnostic or research tools. The invention
surprisingly shows that antibodies may be generated against
exosomes, which recognize these vesicles and do not bind the
exosome-producing cells. Exosome-contacting reagents other than
antibodies consisting of compounds reacting with cognate
exosome-specific biomarkers and that can potentially block the
exosome pathway are also described. Furthermore, the invention
describes methods to identify and characterize cognate
exosome-specific biomarkers. Finally, methods to generate biomarker
derivatives and to use biomarkers and said derivatives as
therapeutic drug to block the exosome pathway are also
described.
[0008] Exosome-contacting reagents that react specifically with
exosomes but not with exosome-producing cells are very much needed
and useful for at least two main purposes. First, the systematic
and accurate characterization of exosomes for research purpose and
clinical use is required. Indeed, reagents used so far to
characterize exosomes are antibodies that recognize known markers
found on cells as well as other cell-derived vesicles.
Exosome-specific reagents will provide a more rigorous means for
the quality control of exosome-based products tested in the
clinics. Second, highly specific exosome reagents represent
therapeutic agents to treat, for instance, pathophysiological
conditions requiring blocking of the exosome pathway.
[0009] The invention relates to a method of generating an
exosome-specific antibody, comprising:
1) preparing vesicles, preferably exosomes from a non-human animal
with a given genetic background; 2) immunizing a non-human animal
with said vesicles, wherein animals for immunization and
preparation of vesicles share the same or very similar genetic
background; and 3) collecting, from said immunized animal,
antibodies reacting with the vesicles of step 1 or corresponding
producing cells.
[0010] The antibodies can be isolated directly from animal serum
for the preparation of polyclonal antibody. Monoclonal antibodies
may also be prepared from these animals using traditional
approaches including generation of hybridomas or isolation of
single antibody-producing cells by methods such as SLAM. In a
specific embodiment, step 3) comprises the steps of a) collecting
lymphocytes from the immunized animal and b) identifying and
isolating single antibody-producing cells producing antibodies
reacting with the vesicles of step 1.
[0011] The specificity of the antibodies may then be assessed in
various assays, particularly binding assays against different
material (e.g., the exosome-producing cell).
[0012] Preferably, the vesicles of step 1) are obtained from
mammalian exosome-producing cells. These cells may be normal,
transformed, tumoral or infected with a virus or microbe. More
preferably, said mammalian exosome-producing cells are murine
cells.
[0013] In another embodiment, the invention relates to a method of
isolating exosomes specific ligands- or reagents, comprising:
1) preparing vesicles, preferably exosomes, 2) screening libraries
of compounds reacting with said vesicles, and 3) selecting exosome
binding ligands from step 2 that do not react with the
exosome-producing cells or with other biological objects,
preferably cells.
[0014] Compound libraries include peptide/polypeptide-, glycolipid-
or nucleotide-based libraries as well as small molecules libraries.
Preferably, the library is a recombinant or natural antibody
library. Non-antibody exosome-contacting compounds may be modified
according to known methods of the art to generate improved exosome
blocking agents. These include notably mutation of binding site or
fusion to partner permitting polymerization, preferably
dimerisation, of exosome-contacting reagent. Such modifications are
expected to increase the affinity of the reagents for exosomes,
thereby improving blocking efficiency. Alternatively,
exosome-contacting reagents may be chemically modified or fused to
partner permitting faster exosome elimination.
[0015] The invention also relates to a method of screening active
compounds, the method comprising contacting in vitro exosomes from
infected cells in the presence of T cells and a candidate compound,
and assessing the ability of the candidate compound to neutralize
infection of T cells. More preferably, the exosomes are obtained
from a virus-infected cell, e.g., an HIV infected cell. Even more
preferably, the exosomes are obtained from infected
macrophages.
[0016] The invention also relates to the use of exosome-specific
ligands to identify exosome-specific biomarkers.
[0017] The invention also relates to a method of identifying
exosome-specific biomarkers comprising:
1) preparing vesicles, preferably exosomes from a non-human animal
with a given genetic background; 2) immunizing a non-human animal
with said vesicles, wherein animals for immunization and
preparation of vesicles share the same or very similar genetic
background; 3) obtaining antibodies reacting with the vesicles of
step 1, and 4) identifying and isolating the cognate biomarkers
using antibodies of step 3.
[0018] Biomarkers identified by this method may be any protein, for
example a receptor or an enzyme, or compounds other than
polypeptides, such as glycolipids, polysaccharides and nucleotides.
Said biomarker may also be a tumor, a viral or a microbial antigen,
depending on the nature of the cells used to produce exosomes for
immunization.
[0019] The invention further concerns the biomarkers targeted by
exosome specific reagents, and their use for research, diagnostic
and therapeutic applications. The invention also concerns a
composition comprising said biomarkers and a pharmaceutically
acceptable excipient or carrier.
[0020] In another embodiment, the invention relates to the
isolation and design of new exosome specific reagents using
exosome-specific biomarkers. Indeed, biomarkers instead of exosomes
may now be used to isolate new exosome-contacting reagents using
the methods described above. Alternatively, because of their
exosome-specificity, biomarkers or their modified forms may
themselves be used as exosome-contacting reagents. Biomarker
modification may include mutagenesis to abolish undesirable
biological activities or increase exosome-contacting affinity,
fusion to polypeptide permitting polymerization, preferably
dimerisation, of the biomarker, fusion to entities improving
exosome elimination.
[0021] The invention also relates to a method of identifying an
exosome-specific biomarker, comprising:
1) preparing vesicles, preferably exosomes from a subject; 2)
injecting the vesicles to the said subject; 3) optionally,
collecting lymphocytes from the subject; and 4) identifying and
isolating single antibody-producing cells producing antibodies
reacting with the vesicles of step 1. 5) preparing the antibodies
produced by antibody-producing cells of step 4 6) identifying and
isolating the cognate biomarkers using antibodies of step 5.
[0022] Preferably, said subject is a patient and exosome-producing
cells are tumor cells or virus or microbe infected cells.
[0023] The invention also relates to a method of producing an
antibody response in a subject against an exosome, comprising:
administering to a subject in need thereof an exosome-specific
biomarker as disclosed above, or as obtained by a method as
disclosed above.
[0024] The invention further concerns isolated exosome-contacting
reagent-producing cells, and their use for reagent production, and
the reagent produced by said cells. The invention also concerns a
composition comprising either said isolated exosome-contacting
reagent-producing cells or said reagent produced by said cells and
a pharmaceutically acceptable excipient or carrier.
[0025] A further object of this invention relates to methods for
neutralizing exosomes in a subject, the method comprising
administering to a subject in need thereof a neutralizing-effective
amount of an exosome-specific ligand, said administration causing a
neutralization of exosomes. The subject may be any mammalian
subject, particularly a human subject. The subject may suffer from
any pathological condition that could benefit from a blocking of
the exosome pathway, including an infectious disease, a cancer, an
autoimmune disease, an inflammatory disease, transplantation,
etc.
[0026] Another object of this invention relates to compositions
comprising an exosome-specific ligand and a pharmaceutically
acceptable carrier or excipient.
[0027] The invention also relates to antibodies, or a fragment or
derivative thereof having substantially the same antigen
specificity, wherein said antibodies selectively bind an exosome.
Specific examples of such antibodies or fragments include any
antibody, or fragment or derivative thereof, which comprises a
heavy chain variable polypeptide sequence encoded by a nucleotide
sequence selected from SEQ ID NOs: 1 to 12. The invention also
encompasses any chimeric polypeptide comprising an antibody, or
fragment or derivative thereof, as defined above.
[0028] A further aspect of this invention resides in
exosome-specific ligands, wherein said ligands comprise an
exosome-specific ligand-binding domain fused to a dimerization or
multimerization domain or compound, in particular to an Fc portion
of an immunoglobulin.
[0029] A further aspect of this invention resides in
exosome-specific ligands, wherein said ligands comprise a protein,
or a domain thereof, that selectively binds an exosome-specific
marker, fused to an exosome-specific antibody or a fragment or
derivative thereof.
[0030] The invention also relates to the use of exosome-specific
ligands to detect exosomes in vitro, ex vivo or in vivo. Detection
may be performed in vitro or ex vivo in any isolated sample, such
as a biological fluid, a tissue, a biopsy, etc. The detection may
also be performed directly in vivo, e.g., using a labeled
exosome-specific ligand.
[0031] The invention further includes methods of characterizing an
exosome preparation, comprising contacting an exosome preparation
with an exosome-specific ligand and determining the presence and/or
amount and/or nature of exosomes in said preparation.
[0032] The invention also relates to the use of an exosome to
stimulate B cells in vitro, ex vivo or in vivo, or to manufacture a
medicament for stimulating a B cell response in a patient.
[0033] The invention also relates to a method of stimulating B
cells in vivo, comprising administering to an animal or a subject
in need thereof an amount of exosomes sufficient to cause a
stimulation of B cells in said animal or subject.
[0034] The invention also relates to a method of stimulating B
cells in vitro, comprising culturing lymphocytes in vitro in the
presence of exosomes, under conditions allowing B cell stimulation.
More preferably, the lymphocytes are obtained from an animal or
subject.
[0035] Preferably, the subject is a patient and the exosomes are
obtained from tumor cells or virus or microbe infected cells.
LEGEND TO FIGURES
[0036] FIG. 1: ELISA detecting anti-Exosome antibody in sera of
exosome-immunized mice.
[0037] FIG. 2: FACS analysis of exosomes and exosome producing
cells with anti-exosome antibodies.
[0038] FIG. 3: Stimulation of B cells by exosomes
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0039] Within the context of this invention, the term "exosome"
refers to externally released vesicles originating from the
endosomic compartment or cells, including tumor cells and immune
cells, particularly antigen presenting cells, such as dendritic
cells, macrophages, mast cells, T lymphocytes or B lymphocytes.
More specifically, such vesicles are of endosomal origin and are
secreted in the extracellular milieu following fusion of late
endosomal multivesicular bodies with the plasma membrane (1).
Methods of producing, purifying or using exosomes for therapeutic
purposes or as research tools have been described in WO99/03499,
WO00/44389 and WO97/05900, incorporated therein by reference.
[0040] An "exosome-contacting reagent" or "exosome-specific ligand
or reagent" designates any compound that binds an exosome and
essentially does not bind the cell from which said exosome derives.
Although non-specific binding to other objects may not be excluded,
such non-specific binding can be discriminated from the specific
binding to exosomes. Exosome-specific ligands or reagents (ESL)
thus typically bind compounds or structure (e.g., biomarkers) that
are specifically or predominantly present at the surface of
exosomes, and essentially absent from other object or cells. ESL
may be derived from diverse classes of natural and synthetic
compounds, including notably antibodies, dimerized cognate ligands
or receptors, small molecules, etc.
[0041] A typical example of ESL includes antibodies, as well as
fragments and derivatives thereof having substantially the same
antigen specificity. Such fragments include Fab, F(ab')2, CDR,
variable regions, etc. Such derivatives include single chain
antibodies, humanized antibodies, human antibodies, bi-functional
antibodies, etc.
[0042] In a specific embodiment of this invention, the ESL is a
monoclonal antibody or a derivative or fragment thereof, even more
preferably an antibody, or fragment or derivative thereof,
comprising a heavy chain variable polypeptide sequence encoded by a
nucleotide sequence selected from SEQ ID NOs: 1 to 12.
[0043] In a further embodiment of this invention, the ESL is a
protein, or a domain thereof, that selectively binds an
exosome-specific marker. A preferred example of such a protein is
lactadherin, or a fragment thereof comprising at least the C1
and/or C2 domain of lactadherin, or a functional equivalent
thereof. Functional equivalents include other proteins or protein
domains containing C1 and/or C2-like domains, such as neuropilin or
del-1, for instance, as well as variants of these proteins, e.g.,
mutants thereof having increased affinity or specificity for
exosomes. Such mutants typically contain 1 or 2 or 3 or 4 or 5
mutated amino acid residues, i.e., replaced, deleted or inserted
within the sequence. The sequence of lactadherin, neuropilin and
del-1 is available from the literature and database.
[0044] The ESL of this invention may be in the form of monomers or
multimers, to further increase their affinity and therefore
specificity towards exosomes. In this regard, in a particular
object, the ESL is a chimeric polypeptide comprising an ESL-binding
domain fused to a dimerization or multimerization domain or
compound, in particular to an Fc portion of an immunoglobulin.
[0045] In a further embodiment of this invention, the ESL comprises
several ESL-binding domains. In this respect, the ESL preferably
comprises a protein, or a domain thereof, that selectively binds an
exosome-specific marker, fused to an exosome-specific antibody or a
fragment or derivative thereof.
[0046] The ESL may be labeled, e.g., using any conventional label
such as fluorescence, luminescence, enzymatic, biochemical or a
radioactive labels.
[0047] The ESL of this invention shall specifically bind exosomes,
as discussed above, thereby allowing exosome detection and
characterization in any sample or in vivo. Furthermore, in a
preferred embodiment, such ESL may be used to neutralize
exosomes.
[0048] In this respect, the term "neutralizing" designates, within
the context of this invention, reducing, blocking, inhibiting or
preventing the activity of exosomes, e.g., their ability to freely
circulate in fluids, to transport and/or present molecules and/or
to efficiently interact with and/or penetrate into cells. Such
neutralization may be partial and temporary. In a preferred
embodiment, the ESL neutralizes exosomes by aggregating these
vesicles, leading to their clearance. Such an aggregation can be
obtained using multimeric or multifunctional ESL, as disclosed
above.
[0049] The present invention discloses antibodies that can be used
to block the exosome pathway and identify exosome-specific
biomarkers. The invention further discloses methods to isolate ESL
other than antibodies and methods to generate biomarker derivatives
that can also be used to block the exosome pathway. The invention
also discloses methods to stimulate B lymphocytes in an animal or
an individual as well as in ex vivo or in vitro cultures. This
invention can be used in experimental, research, therapeutic,
prophylactic or diagnostic areas.
[0050] The present invention stems from the discovery of unexpected
B lymphocyte activation and antibody responses in situations where
the object used for immunization, for instances an exosome, is
derived from cells of identical or similar genetic background than
the non-human animal used for immunization. More particularly, the
invention shows that these antibodies react specifically with
exosomes and thereby identify novel biomarkers of this subcellular
compartment. Generally, antibody responses in animals emanate from
exposure to a foreign or non-self antigen. Antibodies to exosomes
can therefore be generated by immunizing non-human animals in a
xenogenic or allogenic manner. In the first case, the cells
producing exosomes are derived from a different species than that
of the immunized animals. Theoretically, all antigens are foreign
and antibodies against all exosome antigenic entities may be
obtained. In the second case, the cells producing exosomes are
derived from the same species than that of the immunized animals;
however, the two differ in that they express different subtypes of
polymorphic polypeptides. Here, an antibody response restricted
against these polymorphic antigenic entities is expected since all
other antigens are self antigens. As expected, both xenogenic and
allogenic immunization with exosomes yield very strong antibody
responses. By contrast, syngeneic immunization where cells
producing exosomes and immunized animals share identical genetic
background, i.e. cells are derived from the same strain than the
immunized animal, and autologous immunization, where the cells
producing exosomes are derived from the same animal used for
immunization, are not expected to produce any anti-exosome antibody
responses. In this situation, all antigens are recognized as
self-antigens unless the exosome content has been modified to
expose antigenic determinants not present during B cell negative
selection and development process or unless tolerance has been
broken.
[0051] For obvious reasons standard methods for raising antibodies
use xenogenic or allogenic immunization procedures. These responses
are mainly directed against dominant antigenic entities that are
also present in other cell compartments, notably at the surface of
cells. To date, only one antigen, i.e. Lactadherin, appears to be
almost exclusively expressed on exosomes. A domain called C1C2 is
responsible for this specific subcellular distribution and methods
for the preparation of vesicles bearing chimeric proteins that
contain the C1C2 exosome targeting domains and their use are
described in PCT/EP (WO 03/016522). It turns out that earlier
studies suggested that Lactadherin may constitute a potential drug
target in breast cancer (24). More recently, a role of Lactadherin
in stimulating VEGF-mediated vascularization was reported (25).
Hence, Lactadherin may in fact represent the first example of drug
target for anti-exosome antibody therapeutic. However, because
Lactadherin expression is restricted to specific tissues, i.e.
mammary gland (24) and some tumors (our unpublished data), it does
not constitute a true or universal exosome biomarker. In fact,
prior to the instant invention, no antibody reacting with a
non-polymorphic antigenic entity unique to exosomes had ever been
described. The present invention reveals that antibody responses to
exosomes can be induced using a syngeneic immunization procedure
and that this procedure favours raising antibodies directed to
specific new markers on exosomes.
[0052] In addition to the identification of new biomarkers, the
invention presents the advantage of providing new means to
accurately characterize exosome-based pharmaceutical preparations
and potentially, block specifically the exosome pathway.
[0053] Furthermore, the invention also provides a novel method to
stimulate B lymphocytes in vitro and in vivo.
Method for Inducing Exosomes-Specific Antibody Responses
[0054] In this method, antibodies are raised when vesicles,
preferably exosomes, derived from cells of a given genetic
background are introduced into an animal with the same genetic
background than the cells used to prepare exosomes. The production
of antibody may be evaluated by testing the serum of
immunized-animal by standard approaches. The preparation of
antibody may be accomplished by affinity purification of antibodies
from the serum for polyclonal antibodies or by isolating
antigen-specific antibody producing cells for monoclonal
antibodies. Alternatively, monoclonal antibodies may be prepared
using the various known approaches of monoclonal antibody
preparation from immunized animal such as Hybridoma screening (26)
and SLAM (27).
[0055] Immunogens, preferably exosomes, are prepared from cell
cultures and purified by centrifugation on a sucrose gradient (U.S.
Ser. No. 09/780,748). Exosome-producing cells include any cell,
preferably of mammalian origin, that produces and secretes membrane
vesicles of endosomal origin by fusion of late endosomal
multivesicular bodies with the plasma membrane (1,2). Cells from
various tissue types have been shown to secrete exosomes, such as
dendritic cells, B lymphocytes, tumor cells, T lymphocytes and mast
cells, for instance. Methods of producing, purifying or using
exosomes for therapeutic purposes or as research tools have been
described for instance in WO99/03499, WO00/44389, WO97/05900,
incorporated therein by reference. Preferred exosome-producing
cells of this invention are mammalian tumor cells, virus-infected
cells, B and T lymphocytes and dendritic cells, typically of murine
or human origin. In this regard, the cells are preferably
immortalized dendritic cells (WO94/28113), immature dendritic cells
or tumor cells (WO99/03499).
[0056] The cells may be cultured and maintained in any appropriate
medium, such as RPMI, DMEM, AIM V etc., preferably protein-free
media to avoid contamination of exosomes by media-derived proteins.
The cultures may be performed in any suitable device, such as
plates, dishes, tubes, flasks, etc. Alternatively, exosomes may be
prepared from serum of individuals.
[0057] Antibodies are raised following inoculation of exosomes to
animals, preferably in a syngeneic manner, i.e. where immunized
animals have the same genetic background than the cells used to
prepare exosomes. Alternatively, exosomes derived from cells
belonging to the same species but with a different subtype of
polymorphic polypeptides may be used when the expression of the
major polymorphic polypeptides such as MHC I and II molecules is
downregulated. In yet another method, anti-exosomes specific
antibodies may be raised following inoculation of exosomes to
animals in xenogenic or allogenic manner after animals have been
tolerized to foreign or polymorphic polypeptides. Tolerization may
be achieved by method known of the art including repeated exposure
of the animals to the said foreign and polymorphic
polypeptides.
[0058] The antibodies may be polyclonal or monoclonal. Animals can
be from various species, including mice, rodents, primates, horses,
pigs, rabbits, poultry, etc. Preferred animals are mice.
[0059] The inoculum composition generally further comprises a
pharmaceutically acceptable excipient or vehicle, such as a
diluent, buffer, isotonic solution, etc. The composition can
further comprise an adjuvant.
[0060] Administration of inoculum can be performed by various
routes, such as by systemic injection, e.g., intravenous,
intramuscular, intra-peritoneal, intra-tumoral, sub-cutaneous,
intra-splenic, intra-nodal etc.
[0061] The isolation of exosome-specific monoclonal antibodies is
performed by standard methods of antibody repertoire screening,
including ELISA of supernatant from antibody-producing cell
cultures or sorting of cells expressing cell-surface antibodies.
The same exosomes used for immunization is also used as vehicle for
such screening. Antibody-producing cells are preferably primary or
immortalized B lymphocytes. Immortalization may result from
transformation of B lymphocytes with transforming agents including
viruses and oncogenes or from fusion with immortalized cells to
prepare hybridoma. Antibody-producing cells may also be libraries
of prokaryotic or eukaryotic recombinant cells expressing
antibodies, preferably human antibodies. B lymphocytes may be
derived from immunized animals, preferably transgenic animals
expressing human immunoglobulins.
[0062] The invention also relates to the isolation of soluble
antibodies that may be derived from blood of immunized animals or a
fraction thereof. Soluble antibodies may also be derived from any
expression system producing recombinant antibodies individually or
as pools or libraries of recombinant antibodies with various
antigen specificities. Alternatively, other libraries of compounds
that can potentially react with biological objects or molecules,
i.e. peptide/polypeptides, glycolipids, nucleotides or small
molecules may also be used for the screening of ESL. For this
screening, exosome contacting reagents are first isolated by
standard binding procedure, such as affinity-chromatography. ESL
are then selected for not binding to exosome-producing cells in a
second step.
Anti-Exosome Antibodies
[0063] The invention now discloses twelve novel antibodies and
antibody-producing cells that were obtained by the immunization
method described above. These antibodies can potentially be used
for research, diagnostic or therapeutic uses.
[0064] The nucleotide sequences of the variable region of antibody
heavy chains are depicted (SEQ ID 1 to 12). These antibodies were
produced by hybridoma after fusion of the cell line SP2/0 with
spleen cells of mice immunized with syngenic exosomes. Hybridoma
producing these antibodies were identified and isolated by
screening hybridoma supernatants in standard ELISA using exosomes
as antigen and sorting using a fluorescent activated cell sorter
(FACS). The antibodies of the present invention were selected for
the unique properties of reacting with a marker on exosome that is
absent on the surface of the exosome-producing cells. Therefore,
these antibodies identified more likely novel exosome-specific
biomarkers.
[0065] The antibodies of the present invention may be produced
directly from hybridoma. Alternatively, the nucleotide sequences
encoding at least the variable regions of heavy and light chains of
immunoglobulins produced by the hybridomas may be cloned into
expression vectors for their production as full-length or fragments
of recombinant antibodies.
[0066] The invention also relates to a polypeptide composition
comprising at least 50% primary structure identity with the
polypeptides encoded by SEQ ID 1 to 12. Identity may be determined
according to various known techniques, such as by computer
programs, preferably the CLUSTAL method. More preferably, the
polypeptide composition has at least 60% identity, advantageously
at least 70% identity with the polypeptides encoded by SEQ ID 1 to
12. Such polypeptide variant (or functional equivalent) should
retain the ability to react specifically with exosomes and not with
the surface of the producing cells. This property may be verified
as described in the example 2, by comparative FACS staining of both
exosomes and the exosome-producing cells. Possible variations
include amino acid deletion(s), substitution(s), mutation(s) and/or
addition(s). Sequences of antibody and related polypeptide
composition may be further manipulated by recombinant DNA
techniques to increase the affinity of the antibody for its target
antigen, a process known as antibody maturation, or to humanize the
antibody for therapeutic applications. Such variants may also be
produced to design polypeptides with improved pharmacokinetics
properties.
[0067] The invention also concerns a composition comprising either
said isolated antibody-producing cells or said antibodies produced
by said antibody-producing cells and a pharmaceutically acceptable
excipient or carrier.
Exosome-Specific Antigens or Biomarkers
[0068] In an other embodiment, the invention concerns the antigens
or biomarkers targeted by exosome-specific antibodies and their use
for research, diagnostic and therapeutic applications. These
antigens may be non-characterized compounds and antibodies against
said antigens are necessary in order to identify the function of
said antigens and to characterize them. Methods utilizing
antibodies to characterize antigens include notably standard
immunoprecipitation and immunoblotting procedures that enable
antigen isolation. Isolated antigens may then be identified by
various methods known of the art for revealing their composition.
The antigen recognized by the antibodies described above can be any
protein, for example receptors or enzymes, or compound other than
polypeptides, such as glycolipids, polysaccharides and nucleotides
present on exosomes. These antigens may also be novel tumor
antigens, viral antigens, and microbial antigens.
[0069] Obviously, once discovered, exosome-specific biomarkers may
be used in their purified form or in association with exosomes to
identify new ESL using known methods of compound screening. Such
compound may be derived from polypeptides, glycolipids,
polysaccharides, nucleotides or small molecules libraries.
Alternatively, the natural cognate ligands of exosome-specific
biomarkers also constitute new potential ESL. Finally, the
biomarkers themselves or part thereof may become ESL. Indeed,
because of their restricted expression profile to exosomes, it is
expected that biomarkers may contain exosome targeting domains. The
identification of such domain and their usage to target desired
entities to exosomes has been described in PCT/EP (WO 03/016522).
More specifically, the C1C2 domain of Lactadherin mediates the
specific targeting to exosomes of any polypeptide fused to it.
Methods to target antigens, cytokines and other polypeptide to
exosomes and their applications have also been described in PCT/EP
(WO 03/016522). This method can now be used to block the exosome
pathway. For instance, a chimeric construct comprising the C1C2
domain of Lactadherin or its variants fused to the Fc fragment of
an immunoglobulin may have similar consequences than an
anti-exosome antibody for the blocking or elimination of
circulating exosomes.
[0070] Hence the invention concerns the biomarkers or fragments
thereof and their use to either identify new ESL or as part of ESL.
The invention also concerns a composition comprising said
biomarkers and a pharmaceutically acceptable excipient or
carrier.
[0071] Exosome-Pathway Neutralization
[0072] As exosome function unveils, a growing body of information
support that it may be advantageous to block or neutralize the
exosome pathway in specific pathophysiological situations. The
latter include notably cancer and microbial infection with for
instances HIV and HCV. Because of its role in the immune response,
blocking the exosome pathway may also be desirable during
inflammation to restore immune unbalance leading to autoimmunity,
tolerance or immuno-suppression. In this respect, the invention
concerns the use of ESL as therapeutic agents.
[0073] The invention more particularly relates to the use of an
ESL, as defined and disclosed therein, in the preparation of a
pharmaceutical composition for the prevention or treatment of a
pathophysiological condition requiring blocking of the exosome
pathway, including an infectious disease, a cancer, an autoimmune
disease, an inflammatory disease, transplantation, etc.
[0074] In this regard, a particular object of this invention
resides in a method for neutralizing a pathological immune response
in a subject, the method comprising administering to a subject in
need thereof a neutralizing-effective amount of an ESL, said
administration causing a neutralization of said pathological immune
response. The pathological immune response may be selected from an
allergic response, an inflammatory response, including host graft
response following transplantation, and an autoimmune response.
[0075] A particular object of this invention also relates to a
method for neutralizing or treating (viral or microbial) infection
in a subject, the method comprising administering to a subject in
need thereof an amount of an ESL effective to neutralize exosomes
in said subject, said administration causing a neutralization or
treatment of said infection.
[0076] A further particular object of this invention is a method
for neutralizing viral dissemination in a subject, the method
comprising administering to a subject in need thereof an amount of
an ESL effective to neutralize exosomes in said subject, said
administration causing a neutralization of said viral
dissemination.
[0077] As discussed above, the virus may be selected from an
immunodeficiency virus and a hepatitis virus.
[0078] A more specific embodiment of this invention is a method for
neutralizing HIV infection or dissemination in a subject, the
method comprising administering to a subject in need thereof an
amount of an ESL effective to neutralize exosomes in said subject,
said administration causing a neutralization of HIV infection or
dissemination. In a particular embodiment, the method is used in
combination with other anti-viral therapies, e.g., tri-therapy.
[0079] A further particular object of this invention is a method
for treating cancer (e.g., by reducing cancer immune evasion) in a
subject, the method comprising administering to a subject in need
thereof an amount of an ESL effective to neutralize exosomes in
said subject, said administration causing a treatment of said
cancer.
[0080] The ESL or reagent may be an exosome-specific antibody as
described above. Alternatively, they may be biomarker derivatives,
natural cognate ligands of biomarkers or derived from libraries of
compounds other than antibodies also as described above.
[0081] Exosome pathway blocking may be improved by modifying
exosome contacting reagent to either improve their affinity for
their target or permit efficient elimination of exosomes. It could
also lower toxicity or side effects when biomarkers derivatives are
used as blocker. For instance, as mentioned above, Lactadherin is a
biomarker of exosomes that can potentially be used as an ESL to
block the exosome pathway. Lactadherin contains a RGD site
mediating binding to its cognate integrin receptors (24). A point
mutation modifying the RGD site into RGE abolishes this binding and
therefore the biological activity of Lactadherin. Such mutant would
maintain its ability to contact and therefore to potentially block
exosomes. Another modification of Lactadherin could aim at
increasing its affinity for exosomes. This may be achieved for
instances by fusing the C1C2 domain of Lactadherin to the Fc domain
of immunoglobulin. This fusion enables dimerization of Lactadherin
resulting in high binding efficiency. Finally, another example of
modification that may improve the blocking of the exosome pathway
is fusion of two exosome-contacting reagents. For instances, the
heavy chain of the F(ab')2 fragments of antibodies reacting
specifically with exosomes may be fused to the C1C2 domain of
Lactadherin. When such fusion is performed downstream the
dimerization site of immunoglobulin heavy chains, it yields
tetrameric compounds containing two antibody binding sites and two
C1C2 domain of Lactadherin. It is anticipated that the binding
affinity and specificity of this tetrameric reagents will be
greatly increased, thereby improving the efficacy of exosome
pathway neutralization.
[0082] Interference with the exosome pathway or exosome
neutralization using exosome contacting reagents and derivatives as
described above may be provided by injection of the reagents to a
subject.
[0083] In another embodiment the biomarkers may be used in a
purified form or associated with exosomes to induce an antibody
response in a subject. Indeed, biomarker or autologous exosomes
comprising the said biomarker may be injected to a subject to
generate neutralizing anti-exosomes antibodies directly in the said
subject. In this respect, the use of biomarkers or
exosome-containing biomarkers may be therapeutic and also
prophylactic. Exosome-containing biomarkers may be derived from
immortalized or tumor cells. Exosomes may also be derived from
cells infected with viruses or microbes. Exosomes may be derived
from in vitro culture of subject cells or subject body fluid
including serum. Immortalized, tumor or infected cells may be
isolated directly from the subject or prepared in vitro from normal
cells.
B Lymphocyte Stimulation
[0084] The invention now discloses methods to stimulate B
lymphocytes that stem from the unexpected B cell responses obtained
by the immunization method described above. These methods can
potentially be used for research, diagnostic or therapeutic
uses.
[0085] In these methods, B lymphocyte stimulation is induced when
vesicles, preferably exosomes, derived from cells of a given
genetic background are introduced into an animal with the same
genetic background than the cells used to prepare exosomes. Cell
stimulation may be evaluated by assessing the number and frequency
of B lymphocytes in lymphoid organs or in the periphery of
immunized-animal by standard approaches. B lymphocytes may also be
stimulated in vitro when the said vesicles are added to ex vivo
cultures containing B lymphocytes. Cell cultures may comprise mixed
cell populations or purified B lymphocytes cultured as pooled or
single cells. Cell stimulation may be evaluated by measuring
H.sup.3-thymidine incorporation by standard approaches.
[0086] A particular object of this invention is a method of
adjuvant therapy in a subject, the method comprising administering
to a subject in need thereof an amount of an exosome in said
subject, said administration causing the proliferation of B
lymphocytes. Such adjuvant therapy may be performed using exosomes
alone or in combination with an antigen.
[0087] A further particular object of this invention is a method to
expand B lymphocyte cultures in vitro comprising incubating cell
cultures with exosomes, said incubation causing the proliferation
of B lymphocytes. Antigen-specific B cell proliferation may be
achieved by supplementing the cultures with an antigen. Such
stimulation may for instance be used to expand pooled or single
cell cultures containing antibody-producing cells from
immunized-animal or a subject. B lymphocyte proliferation may
facilitate isolating antigen-specific antibody-producing cells
required for the preparation of monoclonal antibodies using the
various known approaches of monoclonal antibody preparation from
immunized animal such as Hybridoma screening (26) and SLAM
(27).
[0088] Further aspects and advantages of the present invention will
be disclosed in the following examples, which shall be considered
as illustrative and not limiting the scope of this application.
EXAMPLES
Example 1
Induction of Anti-Exosome Antibody Responses Upon Syngeneic
Immunization
[0089] The murine lymphoblastoid cell line YAC was selected for
production of exosomes as it is negative for the major polymorphic
markers MHC I and II. Theoretically, most polypeptides on YAC
exosomes are expected to be self antigens regardless of the strain
of mice used for immunization. YAC cells were expanded into 1-liter
spinner flask in ADCF media (Hyclone), a protein-free media for
large-scale production of exosomes. Five-day cell culture
supernatant was transferred into 250-ml centrifuge bottles and spun
5 min at 2000 rpm to pellet cells. The supernatant was then
filtered through 0.2 .mu.m filter and concentrated to 100 ml using
a fiber cartridge with a 500 KD size cut-off. Concentrated
supernatant was then layered onto a 30% sucrose cushion and spun
under 100,000 g for 1 hour 15 min at 4.degree. C. Gradient
interface was collected and sucrose was removed by
PBS-diafiltration using a 500 KD fiber cartridge as above. Multiple
subcutaneous injections of 5 .mu.g of exosomes in PBS were
performed in mice at 15-day intervals. Blood sample were collected
between injections to assess antibody responses. Anti-exosomes
antibodies were detected by standard ELISA using YAC exosomes as
antigen. For the ELISA, 500 ng YAC exosomes in PBS was coated to
the wells of a microtitration plate overnight at 37.degree. C.
Blocking buffer containing 0.05% Tween-20 in PBS was added to the
wells for one hour at room temperature (RT) to saturate the
remaining free binding sites. Wells were then incubated for one
hour at RT with serum of immunized mice at a dilution 1/500 in
Blocking buffer. After washing the wells three times with Blocking
buffer, bound antibodies were detected using a 1/10000 dilution of
horse-radish peroxidase (Jackson ImmunoResearch) and an ECL
substrate (Amersham). The results of this ELISA are shown in FIG.
1.
[0090] Results: A signal above background was detected after
multiple immunizations with exosomes (1-1 to 1-3). Background was
determine as the signal detected when using serum collected
pre-immunization (P-I).
[0091] Conclusion: Although the exosomes used for immunization are
expected to contain only self antigens, an antibody response was
detected. Despite the multiple injections and the large quantity of
material injected, the titer of anti-exosome antibodies in the
serum of immunized mice remains very low. This response profile is
in line with the non-classical immunization procedure used in the
present invention.
Example 2
Specificity of Anti-Exosome Antibodies
[0092] Spleen cells of mice immunized as described in Example 1
were fused to SP2/0 to produce hybridoma. Hybridomas were then
screened and selected for production and secretion of anti-exosome
antibodies by ELISA also as described in Example 1. Antibodies of
the IgM subtype produced by the Hybridoma clone 45, 101 and 62
(negative control) were purified from culture supernatant by
diafiltration using a fiber cartridge with a 500 KD size cut-off.
The purity of diafiltered and concentrated fractions was verified
to be at least 90% by SDS-PAGE analysis. The purified antibodies
were used to stain exosomes coated on 1-.mu.m beads as well as
exosome-producing cells. Staining was detected by FACS analysis
using a secondary anti-mouse IgM conjugated to a fluorophore. The
results of this analysis are shown in FIG. 2.
[0093] Results: A shift of fluorescence was detected when YAC
exosome-coated beads were incubated with IgM 45 and 101, whereas no
fluorescence was detected with the negative control antibody IgM 62
(FIG. 2, panel A). In contrast, IgM 45 and 101 did not bind to YAC
cells as cell staining did not yield a fluorescent shift with any
antibody tested (FIG. 2, panel B). Furthermore, IgM 45 and 101 were
also found to react with exosomes from a different cell type and
species since a shift of fluorescence was detected with these
antibodies when beads were coated with human dendritic cell-derived
exosomes or dexosomes (FIG. 2, panel 3).
[0094] Conclusion: This data suggest that IgM 45 and 101 recognize
antigenic determinant(s) expressed on the exosomes used for
immunization but not on the surface of the cells used to produce
the said exosomes. Moreover, this(these) antigenic determinant(s)
is(are) conserved in exosomes derived from different cell types and
species. Hence they most likely represent novel entities or
biomarkers specific to the exosome compartment.
Example 3
In Vivo and In Vitro Stimulation of B Cells by Exosomes
[0095] Spleen and popliteal lymph nodes of mice immunized as
described in example 1 were collected and cells were counted using
an hematocytometer and stained with PE-conjugated anti-CD19
antibody to determine the percentage of B cells. Cells were also
plated into the wells of 96-well tissue culture plated at
2.times.10E5 cells/wells in RPMI/15% FCS supplemented or not with
exosomes. Spleen cell samples depleted of CD19-positive cells using
Myltenii magnetic beads were also used in the assay. Following a
3-day incubation at 37.degree. C./5% CO.sub.2, 1 .mu.Ci
H.sup.3-thymidine was added to each well and proliferation was
counted 16 hours later. The assay was performed in triplicate and
the results, shown in FIG. 3, are reported as Stimulation Index
over the mean count obtained with cells of unstimulated naive
mice.
[0096] Results: Based on counts from about ten experiments using at
least 3 animals per experiment, the number of cells in PLN of naive
mice was on average 5.times.10.sup.5 to 1.times.10.sup.6 cells per
node compared to 1.5.times.10E6 to 8.times.10E6 cells per node in
exosome-immunized mice. This increased number in cells was not
observed in spleen cells, most likely because of the route of
immunization used. FACS analysis of PLN cells revealed an average
of 25.5+/-4.2% CD19+ cells in naive PLN whereas 40+/-3.6% of the
same cells was detected in exosome-immunized mice. Furthermore, as
shown FIG. 3 panel A, PLN cells from exosome-immunized mice (1-PLN)
were activated as indicated by their potency to proliferate even in
the absence of stimulation in vitro. Both PLN cells from naive
(N-PLN) and immunized mice were stimulated by incubation with YAC
exosomes. Higher SI was obtained with I-PLN cells more likely
because these cells are already activated compared to the cells
from the N-PLN group. This effect was exosome species specific
since incubation with exosome derived from a human cell line (221)
did not lead to proliferation above the level of unstimulated
cells. This proliferation profile could also be detected with
spleen cells (FIG. 3, panel B) although the stimulation indexes
were much lower with these cells. Again, this is most likely
because of the route of immunization used. Nevertheless,
proliferation was abolished in all stimulation groups when
CD19-depleted cells were used (1-S/CD19-) indication that B cell
proliferation was measured in this assay.
[0097] Conclusion: Injection of YAC exosomes in mice induced
activation of B cells in PLN and spleen. Naive B cells could also
be induced to proliferate when incubated with YAC exosomes but not
human-derived exosomes. Therefore, YAC exosomes display adjuvant
properties and may be used in vivo and in vitro to stimulate B
cell.
REFERENCES
[0098] 1. Thery C, Zitvogel Z, Amigorena S. Exosomes: composition,
biogenesis and function. Nat. Rev. Immunol. 2(8), 569-579 (2002).
[0099] 2. Delcayre A., Shu H., Le Pecq J. B. Dendritic cell-derived
Exosomes in Cancer Immunotherapy: Exploiting Nature's Antigen
Delivery Pathway. Expert Rev. Anti-Cancer Therapy 5(3), in press
(2005). [0100] 3. Zitvogel L, Regnault A, Lozier A et al.
Eradication of established murine tumors using a novel cell-free
vaccine: dendritic cell-derived exosomes. Nat. Med. 4(5), 594-600
(1998). [0101] 4. Morse M A, Garst J, Osada T, et al. A phase I
study of dexosome immunotherapy in patients with advanced non-small
cell lung cancer. J. Transl. Med. 3(1), 9 (2005). [0102] 5.
Escudier B, Dorval T, Chaput N et al. Vaccination of metastatic
melanoma patients with autologous dendritic cell (DC) derived
exosomes: results of the first phase I clinical trial. J. Transl.
Med. 3(1), 10 (2005). [0103] 6. Wolfers J, Lozier A, Raposo G et
al. Tumor-derived exosomes are a source of shared tumor ejection
antigens for cross-priming. Nat. Med. 7(3), 297-303 (2001). [0104]
7. Andre F, Schartz N E, Chaput N, Flament C, Raposo G, Amigorena
S, Angevin E, Zitvogel L. Tumor-derived exosomes: a new source of
tumor rejection antigens. Vaccine 20(Suppl 4), A28-31 (2002).
[0105] 8. Andre F, Schartz N E, Movassagh M, Flament C, Pautier P,
Morice P, Pomel C, Lhomme C, Escudier B, Le Chevalier T, Tursz T,
Amigorena S, Raposo G, Angevin E, Zitvogel L. Malignant effusions
and immunogenic tumour-derived exosomes. Lancet 360(9329), 295-305
(2002). [0106] 9. Altieri S L, Khan A N, Tomasi T B. Exosomes from
plasmacytoma cells as a tumor vaccine. J. Immunother. 27(4), 282-8
(2004). [0107] 10. Taylor D D, Gercel-Taylor C. Tumour-derived
exosomes and their role in cancer-associated T-cell signalling
defects. Br. J. Cancer. 92(2), 305-11 (2005). [0108] 11. Peche H,
Heslan M, Usal C, Amigorena S, Cuturi M C. Presentation of donor
major histocompatibility complex antigens by bone marrow dendritic
cell-derived exosomes modulates allograft rejection.
Transplantation 76(10), 1503-10 (2003). [0109] 12. Andreola G,
Rivoltini L, Castelli C, Huber V, Perego P, Deho P, Squarcina P,
Accornero P, Lozupone F, Lugini L, Stringaro A, Molinari A, Arancia
G, Gentile M, Parmiani G, Fais S. Induction of lymphocyte apoptosis
by tumor cell secretion of FasL-bearing microvesicles. J. Exp. Med.
195(10), 1303-16 (2002). [0110] 13. Frangsmyr L, Baranov V, Nagaeva
O, Stendahl U, Kjellberg L, Mincheva-Nilsson L. Cytoplasmic
microvesicular form of Fas ligand in human early placenta:
switching the tissue immune privilege hypothesis from cellular to
vesicular level. Mol. Hum. Reprod. 11(1), 35-41 (2005). [0111] 14.
Janowska-Wieczorek A, Wysoczynski M, Kijowski J, Marquez-Curtis L
Machalinski B, Ratajczak J, Ratajczak M Z. Microvesicles derived
from activated platelets induce metastasis and angiogenesis in lung
cancer. Int. J. Cancer. 113(5), 752-60 (2005). [0112] 15. Hugel B,
Martinez M C, Kunzelmann C, Freyssinet J M. Membrane
microparticles: two sides of the coin. Physiology (Bethesda) 20(1),
22-27 (2005). [0113] 16. Whiteside T L. Tumour-derived exosomes or
microvesicles: another mechanism of tumour escape from the host
immune system? Br. J. Cancer 92(2), 209-11 (2005). [0114] 17. Gould
S J, Booth A M, Hildreth J E. The Trojan exosome hypothesis. Proc.
Natl. Acad. Sci. USA 100(19), 10592-7 (2003). [0115] 18. Nguyen D
G, Booth A, Gould S J, Hildreth J E. Evidence that HIV budding in
primary macrophages occurs through the exosome release pathway. J.
Biol. Chem. 278(52), 52347-54 (2003). [0116] 18b. Sharova et al.,
the EMBO J. (2005) 1-9. [0117] 18c. Pelchen-Matthews et al.,
Journal of Cell Biology 162 (2005) 443-455 [0118] 19. Morita E,
Sundquist W I. Retrovirus budding. Annu. Rev. Cell Dev. Biol. 20,
395-425 (2004). [0119] 20. Masciopinto F, Giovani C, Campagnoli S.
et al. Association of hepatitis C virus envelope proteins with
exosomes. Eur. J. Immunol. 34(10), 2834-42 (2004). [0120] 21.
Pelchen-Matthews A, Raposo G, Marsh M. Endosomes, exosomes and
Trojan viruses. Trends Microbiol. 12(7), 310-6 (2004). [0121] 22.
Flanagan J, Middeldorp J, Sculley T. Localization of the
Epstein-Barr virus protein LMP 1 to exosomes. J. Gen. Virol. 84,
1871-9 (2003). [0122] 23. Gutwein P, Stoeck A, Riedle S et al.
Cleavage of 11 in exosomes and apoptotic membrane vesicles released
from ovarian carcinoma cells. Clin Cancer Res. 11 (7), 2492-501
(2005). [0123] 24. Taylor M R, Couto J R, Scallan C D, Ceriani R L,
Peterson J A. Lactadherin (formerly BA46), a membrane-associated
glycoprotein expressed in human milk and breast carcinomas,
promotes Arg-Gly-Asp (RGD)-dependent cell adhesion. DNA Cell Biol.
16(7):861-9 (1997). [0124] 25. Silvestre, J. S., Thery, C., Hamard,
G., Boddaert, J., Aguilar, B., Delcayre, A., Houbron, C., Tamarat,
R., Clergue, M., Duriez, M., Merval, R., Levy, B., Tedgui, A.,
Amigorena, S, and Mallat, Z. Lactadherin/MFG-E8: a novel angiogenic
protein required for VEGF signalling. Nature Med. 11(5), 499-506
(2005). [0125] 26. Harlow E and Lane D. Antibodies: A laboratory
manual. Cold Spring Harbor Laboratory, (1988). [0126] 27. Babcook,
J. S.; Leslie, K. B.; Olsen, O. A.; Salmon, R. A. and Schrader, J.
W. A novel strategy for generating monoclonal antibodies from
single, isolated lymphocytes producing antibodies of defined
specificities. Proc. Natl. Acad. Sci. U.S.A 93,7843-48 (1996).
Sequence CWU 1
1
121343DNAArtificialNucleotide sequence encompassing the heavy chain
variable region of immunoglobulin produced by hybridoma 2.1
1gctcccaggn caactccagc agcctggggc tgaaactggt gaagcctggg gcttcagtga
60agttgtcctg caaggcttct ggctacacct tcaccagcta ctggatgcac tgggtgaagc
120tgaggcctgg acaaggcttt gagtggattg gagagattaa tcctagcaat
ggtggtacta 180actacaatga gaagttcaag agaaaggcca cactgactgt
agacaaatcc tccancacag 240cctacatgca accagcaacc tgacatctga
ggactctgcg gtcnattact gtacaatcct 300caangcttac tggggccaan
ggactcnggt cacagtctct gca 3432367DNAArtificialNucleotide sequence
encompassing the heavy chain variable region of immunoglobulin
produced by hybridoma 6.1 2ggggcagagc ttgtgaggtc aggggcctca
gtcaagttgt cctgcacagc ttctggcttc 60aacattaaag actactatat gcactgggtg
aagcagaggc ctgaacaggg cctggagtgg 120attggatgga ttgatcctga
gaatggtgat actgaatatg ccccgaagtt ccagggcaag 180gccactatga
ctgcagacac atcctccaac acagcctacc tgcagctcag cagcctgaca
240tctgaggaca ctgccgtcta ttactgtaat gcatgggggt actttgacta
ctggggccaa 300ggcaccactc tcacagtctc ctcagagagt cagtccttcc
caaatgtctt ccccctcgtc 360tcctgca 3673334DNAArtificialNucleotide
sequence encompassing the heavy chain variable region of
immunoglobulin produced by hybridoma 19.2 3ggactgactg gtaaggcctg
ggacttcagt gaagatatcc tgcaaggctt ctggctacac 60cttcactaac tactggctag
gttgggtaaa gcagaggcct ggacatggac ttgagtggat 120tggagatatt
taccctggag gtggttatac taactacaat gagaagttca agggcaaggc
180cacactgact gcagacacat cctccagcac tgcctacatg cagctcagta
gcctgacatc 240tgaggactct gctgtctatt tctgtgcaag atggtggtta
cgacgggaat ggtttgctta 300ctggggccaa gggactctgg tcactgtctc tgca
3344340DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 20.1
4ggggctgaac tggcaagcac ctggggcctc agtgaagatg tcctgcaagg cttctggcta
60cacctttact agctacacga tgcactgggt aaaacagagg cctggacagg gtctggaatg
120gattggatac attaatccta gcagtggtta tactaattac aatcagaagt
tcaaggacaa 180ggccacattg actgcagaca aatcctccag cacagcctac
atgcaactga gcagcctgac 240atctgaggac tctgcagtct attactgtgc
aagaggggga ctattatatt actatgctat 300ggactactgg ggtcaaggaa
cctcagtcac cgtctcctca 3405352DNAArtificialNucleotide sequence
encompassing the heavy chain variable region of immunoglobulin
produced by hybridoma 45-1 5ggagtcagga cctggcctgg tggcaccctc
acagagcctg tccatcacat gcactgtctc 60tgggttctca ttatccagat atagtgtaca
ctgggttcgc cagcctccag gaaagggtct 120ggagtggctg ggaatgatat
ggggtggtgg aagcacagac tataattcag ctctcaaatc 180cagactgagc
atcagcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct
240gcaaactgat gacacagcca tgtactactg tgccagaacc tctctctact
ataggtacgg 300tcctagagct atggactact ggggtcaagg aacctcagtc
accgtctcct ca 3526334DNAArtificialNucleotide sequence encompassing
the heavy chain variable region of immunoglobulin produced by
hybridoma 47.1 6ggaagaagct tggtgcaacc tggagggatc catgaaactc
tcctgtgttg cctctggatt 60cactttcagt aactactgga tgaactgggt ccgccagtct
ccagagaagg ggcttgagtg 120ggttgctgaa attagattga aatctaataa
ttatgcaaca cattatgcgg agtctgtgaa 180agggaggttc accatctcaa
gagatgattc caaaagtagt gtctacctgc aaatgaacaa 240cttaagagct
gaagacactg gcatttatta ctgttctgat ggttactacc cgtttgctta
300ctggggccaa gggactctgg tcactgtctc tgca
3347332DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 52.1
7agcttactga acctggaggg tccctgaaac tctcctgtgc agcctctgga ttcgctttca
60gtagctatga catgtcttgg gttcgccaga ctccggagaa gaggctggag tgggtcgcaa
120ccattagtag tggtggtagt tacacctact atccagacag tgtgaagggc
cgattcacca 180tctccagaga caatgccagg aacaccctgt acctgcaaat
gagcagtctg aggtctgagg 240acacggcctt gtattactgt gcaagacaga
cagccaaggt ccttgcctgg tttgcttact 300ggggccaagg gactctggtc
actgtctctg ca 3328343DNAArtificialNucleotide sequence encompassing
the heavy chain variable region of immunoglobulin produced by
hybridoma 60.1 8ggaccgaaat ggtcggcctg ggacttcagt gaaggtgtcc
tgcaaggctt ctggatacgc 60cttcactaat tacttgatan agtgggtaaa gcagaggcct
ggacagggcc ttgagtggat 120tggagtgatt aatcctggaa gtggtggtac
taactacaat gagaagttca agggcaaggc 180aacactgact gcagacaaat
cctccagcac tgcctacatg cagctcagca gcctgacatc 240tgatgactct
gcggtctatt tctgtgcaag agcctatagg tacnactgag gctactggta
300cttcgatgtc tggggcgcag ggaccacggt caccgtctcn tca
3439316DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 61.6
9ggactgagct ggtgaacctg gggcttcagt gaagatatcc tgcaagactt ctggatacac
60attcactgaa tacaccatgc actgggtgaa gcagagccat ggaaagagcc ttgagtggat
120tggaggtatt aatcctaaca atggtggtac tagctacaac cagaagttca
agggcaaggc 180cacattgact gtagacaagt cctccagcac agcctacatg
gagctccgca gcctgacatc 240tgaggattct gcagtctatt actgtgcaag
actgggacgc tactggggcc aaggcaccac 300tctcacagtc tcctca
31610328DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 62.8
10ggactgagct ggtgaacctg gggcttcagt gaagatgtcc tgtaaggctt ctggatacac
60attcactgac tactacatga agtgggtgaa gcagagtcat ggaaagagcc ttgagtggat
120tggagatatt aatcctaaca atggtggtac tagctacaac cagaagttca
agggcaaggc 180cacattgact gtagacaaat cctccagcac agcctacatg
cagctcaaca gcctgacatc 240tgaggactct gcagtctatt actgtgcaag
agatacggcc tggtacttcg atgtctgggg 300cgcagggacc acggtcaccg tctcctca
32811356DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 101.5
11ggggcctcag tgaacatatc ctgcagggct actggctaca ctttcgataa ctactggata
60gagtgggtaa agcagaggcc tggacatgcc cttgagtgga ttggagacat tttacctgga
120agtggtacta ctaactacaa tgagaaattc aggggcaagg ccacgttcac
tgcacatacg 180tcctccaaca caccctaaat gcaactcagc aacctgacat
ccgaggactc tgccttctat 240tactgtgcac ggcattacta cggtagtacc
tcatttgctt actggggcca agggactctg 300gtccctgtct ctgcagagag
tcactccttc ccaagtgtct tccccctcgt ctcctg
35612371DNAArtificialNucleotide sequence encompassing the heavy
chain variable region of immunoglobulin produced by hybridoma 107.1
12ggagtcagga cctggcctgg tggcgccctc acagagcctg tccatcactt gcactgtctc
60tgggttttca ttaaccagct atggtgtaca ctgggttcgc cagcctccag gaaagggtct
120ggagtggctg ggagtaatat gggctggtgg aagcacaaat tataattcgg
ctctcatgtc 180cagactgagc atcagcaaag acaactccaa gagccaagtt
ttcttaaaaa tgaacagtct 240gcaaactgat gacacagcca tgtactactg
tgccagaggc ggaggctatg gtaactacgg 300gtactggggc caagggactc
tggtcactgt cnctntagag antcagtcct tcccaaatgt 360cttccccctc g 371
* * * * *